Project Summary We will continue to design, construct and characterize genetic circuits. We will use micro uidic tools to grow and observe single cells and colonies in precisely controlled environmental conditions, and we will test the engineered bacterial strains in tumor spheroids. We will characterize circuit-host interactions and develop new design principles. The characterization of cellular behavior across multiple experimental platforms will inform mathematical models that will be used to identify key design characteristics, which will then be rigorously tested using previously established techniques. Two Postdocs, two Sta Research Scientists, and a Graduate Student Researcher will work with Drs. Hasty and Tsimring on multiple aspects of the project in an integrated manner. Our track record demonstrates our ability to train personnel in a multi-disciplinary approach that has led to new tools for synthetic biology, along with an increased understanding of gene and signaling networks generally. Our recent characterization of bacterial circuits in animal models has served to highlight the need for a better understanding of how engineered bacteria function in a tumor environment. Accordingly, our Speci c Aims focus on the development of delivery circuits in small ecologies (Aim 1), the characterization of engineered bacteria in tumor spheroids (Aim 2), and the interaction of circuits with their hosts (Aim 3). Our rst aim is to develop small ecological delivery systems consisting of bacterial strains that can be found in the tumor environment. One system will generate regular out-of-phase delivery sequences, while a second will be designed for chaotic dynamics that could be useful for therapies that evade tumor adaptation. We will develop computational models and experimentally quantify how the circuits behave in micro uidic devices. While such mathematical models are generally e ective in predicting the population dynamics of engineered bacteria when grown in isolation, the complex environment of a tumor does not represent a simple extension of our existing understanding. The experimental cycle for animal models is too long and costly for the development of an engineering-based approach to circuit design. Our second aim will be to use a tumor spheroid platform for the development of mathematical modeling for engineered bacteria that reside in tumors. We will use the ndings to identify essential modi cations to the computational modeling. Finally, gene circuits are typically engineered with model equations that assume isolation from the host. The third aim will combine integrative circuit-host modeling with a high-throughput micro uidic platform to quantitatively characterize the bidirectional coupling between engineered gene circuits and their hosts. We will explore the e ects of environmental constituents that are present in sold tumors and evaluate the circuit-genome response to tumor spheroid lysate (from Aim 2). The goal of this aim is to deduce fundamental principles that improve the design-build-test-re ne process for gene circuits.